It is well established that extracts obtainable from plants of the Asclepiadaceae family, particularly the Hoodia genus (formerly the Hoodia and Trichocaulon genera) have been shown to have an appetite suppressant activity associated with specific steroidal glycosides. U.S. Pat. No. 6,376,657 discloses that these plants contain steroidal glycosides according to Formula 1:
wherein:R=alkyl;R1=H, alkyl, tigloyl, benzoyl or any other organic ester group;R2=H or one or more 6-deoxy carbohydrates, or one or more 2,6-dideoxy carbohydrates, or glucose radicals or combinations thereof;and wherein the broken lines indicate the optional presence of a further bond between carbon atoms C4 and C5 or between carbon atoms C5 and C6.
U.S. Pat. No. 6,376,657 also discloses processes to extract steroidal glycosides according to Formula 1 from Hoodia plants, which involves treating plant material with a solvent to extract a fraction having appetite suppressant activity, separating the extraction solution from the rest of the plant material, removing the solvent from the extraction solution, optionally treating the solution with the additional solvent, and recovering the extract. The solvents specifically disclosed include methylene chloride, also known as dichloromethane. The patent also discloses methods for synthesizing various steroidal glycosides.
WO2005/116049 discloses that steroidal glycosides can be extracted or separated from undesirable components present in plant material of the Asclepiadaceae family by means of liquid or supercritical carbon dioxide extraction.
However, the use of a plant extract derived from an intact plant or parts of an intact plant has a number of disadvantages. Firstly, sufficient amounts of plant material have to be obtained for the extraction process. Harvesting is limited to a very few countries where the correct natural conditions are present to allow Hoodia to grow naturally. Outside of these countries, Hoodia can only be grown in specialised conditions in a greenhouse with great difficulty. Furthermore, Hoodia plants have a long life cycle and therefore, growing large quantities of plant material is not only expensive but also time consuming.
It is well known in the art that plant cells can be maintained and grown in tissue culture. Tissue culture is a term used in the art to define the growth of plant cells outside an intact plant in a suitable nutrient medium. Tissue culture is defined as a method wherein parts of a plant are transferred to an artificial environment in which they can continue to survive. The term tissue culture as understood in the art refers to cultured tissue which may consist of individual or groups of plant cells, protoplasts or whole or parts of a plant organ.
In tissue culture, plant cells can be grown on a solid surface as or as individual or small clusters of cells in suspension cultures. Cells grown in culture are actively dividing and can be maintained indefinitely in an undifferentiated state by transferring the cells to fresh media (sub-culturing). Cultured cells can also be induced to re-differentiate into whole plants. It has been shown that whole plants regenerated from callus cultures produce genetic variants.
Tissue culture is a method well known in the field of plant biology and has several applications, for example it is used to produce large quantities of plants or plant material by vegetative multiplication in a short period of time (micro-propagation). Plant tissue cultures can be initiated from almost any part of the source plant (termed explant), although younger parts of a plant are generally more useful as they contain a higher amount of dividing cells. Although tissue culture is a method well known in the art, different plants may vary in the exact conditions for maintaining cells in culture. Cells in tissue culture are generally different from in vivo cells (cells in an intact plant which have not been isolated from the plant and cultured), for example they have a small vacuole, lack chloroplasts and photosynthetic pathways. It is also well known in the art that cultured plant cells produce different amounts and altered profiles of metabolites.
One way of tissue culturing plant cells is by suspension culture, which is disclosed in WO2006/051334. In a suspension culture, small clusters of cells are grown in a flask suspended in a culture media. The culture or nutrient media typically comprise carbohydrates as a source of energy, salts, vitamins, amino acids, minerals, plant growth hormones and other compounds. The flasks containing the cells and the culture media are typically stored on a shaker to prevent the cells from settling at the bottom of the flask. Suspension cultures are sub-cultured at specified intervals, for example every three weeks, to provide fresh growth media and to maintain the cells in an undifferentiated state. The cells obtained are treated by freeze drying, spray drying, vacuum drying or homogenisation techniques following culturing in order to change the physical nature of the cells. The cells can further be treated with one or more solvents in order to collect an extract fraction comprising at least one compound having appetite suppressant activity.
However, a significant problem with such techniques is that the use of solvents, such as alcohols, can denature the active ingredients (steroidal glycosides) within the plant extracts. The need remains therefore for alternate processes of obtaining therapeutically useful materials having a high content of steroidal glycosides which are suitable for use in foods, beverages or supplements.
EP2329836 A1 purports to disclose a process for the preparation of a plant extract from Hoodia gordonii suitable for use as an appetite suppressant. However, upon detailed examination of the document it is clear that the disclosure of EP23289836 is deficient and any extract produced according to the processes disclosed in EP2329836 will not actually contain therapeutically significant amount of steroidal glycosides.
It is well known that the steroidal glycosides in Hoodia gordonii are generated within the plant to protect it from UV damage. In the absence of a UV light source only a relatively small amount of steroidal glycosides is generated. For example, U.S. Pat. No. 4,562,250 discloses that much greater quantities of steroidal glycosides are generated in Yucca cell cultures that grown in light than are grown in darkness. EP2329836 discloses four alternative methods of preparing Hoodia gordonii plant extracts: examples 1 to 4 on page 6 of the specification. In each of these examples a cell line of Hoodia gordonii is grown in the dark at a temperature of 25° C. for 14 days. Crucially, all of the cell lines are grown in the dark without any light source. It is an inevitable outcome of being grown in the dark that the resulting cells will contain only relatively low levels of steroidal glycosides.
As the extracts of EP2329836 contain only low levels of steroidal glycosides it must be assumed that the demonstrated appetite suppressing and fungicidal effects of those extracts cannot be due to therapeutic levels of steroidal glycosides in the extracts. Rather, any such effects must be due to the presence of other chemicals or components in the extracts, for example solvents that are used to extract the steroidal glycosides.
Tangential flow (or crossflow) filtration technique (TFF) is an effective method to separate or remove cell debris, and recover biosynthetic substances after bacterial fermentation (‘Separation of hyaluronic acid from fermentation broth by tangential flow microfiltration and ultrafiltration’, Zhou et al, Separation and Purification Technology (2006), No 52, pp. 29-38). In TFF, bulk flows tangentially across a membrane and perpendicularly to a permeation flux. The filtering performance of TFF depends on the properties of the membrane, the product and operational conditions such as transmembrane pressure, crossflow velocity, solution chemistry, concentration factor and running time etc. TFF is usually considered to be effective on the condition that solutes differ by more than a 10-fold difference in size. High performance TFF (HPTFF) has also been developed to achieve separation of solutes differing by less than a 10-fold difference in size according to Zhou et al.
In light of all of the above there is need for an improved method of obtaining a medium containing steroidal glycosides that is suitable for therapeutic use as an appetite suppressant or for any other appropriate use.